The overall goal of the four interdependent projects in this Program Project is to carry out functional genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has become a model for the assemblage of over 250,000 species of non-yeast fungi. Most Neurospora genes have no homologs in yeasts and nearly 40% have no strong homologs in any organism to date, suggesting that examination of the functions of these genes will both novel and informative. Neurospora is an important model for basic research in eukaryotes, and fungi allied to Neurospora include significant animal and plant pathogens and industrial strains yielding antibiotics, chemicals, enzymes, and pharmaceuticals. This subproject will pursue the systematic disruption of genes through targeted gene replacements, preliminary phenotyping of these strains, and their distribution to the scientific community at large. The 43 Mb Neurospora genome is completely sequenced, and automated annotation using programs trained on Neurospora genes predict 10,082 proteins. Phenotypes are now associated with about 10% of these, and roughly half of the genes have no strong sequence homologs in other organisms; thus, there are no clues to their function(s). Furthermore, it is likely that functions of some genes sharing sequence similarity with genes in other organisms will be modified slightly or greatly, reflecting the novel biology of filamentous fungi. Our long term goal is to create gene knockouts in all of these genes as a first step to determining each gene's function(s), and the immediate goal of the five Specific Aims of Project #1 is to facilitate this. In Specific Aim #1, we will use PCR, homologous recombination in yeast, and long range PCR to generate constructions (knockout cassettes) that can be used to knockout all of the genes in Neurospora. In Specific Aim #2, we will carry out gene replacements in Neurospora to generate unambiguous functional knockouts. In Specific Aim #3, we will carry out a basic phenotypic characterization of all the mutants. In Specific Aim #4, we will deposit the novel strains in the Fungal Genetics Stock Center which will maintain the stocks and distribute them to the scientific community at large. This effort will help to anchor genomic exploration in the Kingdom of the Fungi.